Three new prenylated xanthones from Comastoma pedunculatum and their anti-tobacco mosaic virus activity

Phytochemistry Letters 11 (2015) 245–248

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Three new prenylated xanthones from Comastoma pedunculatum and their anti-tobacco mosaic virus activity Min Zhou a,b, Kun Zhou a, Ying-Liang Zhao b, Neng-Jun Xiang b, Tian-Dong Zhang b, Yue-De Wang a, Wei Dong a, Bing-Kun Ji a, Li-Mei Li a, Jie Lou a, Gan-Peng Li a,*, Qiu-Fen Hu a,** a

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650031, PR China b Technology Center, China Tobacco Yunnan Industry Company (Ltd.), Kunming 650000, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 November 2014 Received in revised form 5 January 2015 Accepted 6 January 2015 Available online 15 January 2015

Comastomaxanthones A–C (1–3), three new xanthones with a rare 2-oxoprenyl substituent, along with three known ones (4–6) were isolated from the aerial parts of Comastoma pedunculatum. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. In addition, compounds 1–6 were tested for their anti-tobacco mosaic virus activity. The results showed that all isolated compounds exhibited weak anti-TMV activity with IC50 values ranging from 122.7 to 242.9 mM. ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Xanthones Comastoma pedunculatum Anti-tobacco mosaic virus activity

1. Introduction The herbs of Comastoma pedunculatum (Royle ex D. Don) Holub (Gentianaceae), a traditional Tibetan medicine named ‘‘Zangyinchen’’, have been widely used for treatment of hepatitis, liver fibrosis, and cholecystitis by local inhabitants in the Tibetan area for centuries (Qiao et al., 2012; Tang et al., 2011). Due to their potential beneficial pharmacological effects, much attention has been paid to the search for biological constituents from this plant. Previous phytochemical investigations of C. pedunculatum have uncovered a series of structurally diverse metabolites, such as xanthones, flavonoids, lignins, and triterpenoids (Qiao et al., 2012; Tang et al., 2011, 2013). Among them, xanthones are considering as the characteristic chemical constituents of this plant, which exhibit a wide range of biological properties, including anticancer, antiviral, antimicrobial, anti-inflammatory, and antioxidant activities (El-Seedi et al., 2010; Mahabusarakam et al., 2008; Wu et al., 2013; Vo et al., 2012). In search for new bioactive compounds from

* Corresponding author. ** Corresponding author. Tel.: +86 871 5913043. E-mail addresses: [email protected] (G.-P. Li), [email protected] (Q.-F. Hu).

traditional Chinese medicines, the chemical composition of the aerial parts of C. pedunculatum was systematically investigated herein. Three previously unreported xanthones with a rare 2oxoprenyl group, named comastomaxanthones A–C (1–3), were isolated along with three known ones (4–6). In this paper, the structure elucidation of compounds 1–3, and the anti-tobacco mosaic virus (anti-TMV) activities of the isolated compounds, are reported. 2. Results and discussion Repeated column chromatography over silica gel, semipreparative and preparative HPLC of the acetone extract from the aerial parts of C. pedunculatum, led to the isolation of three new xanthones with a rare 2-oxoprenyl group, named comastomaxanthones A–C (1–3), along with three known ones (4–6) (Fig. 1). Compound 1, obtained as a yellow gum, was determined to have the molecular formula C22H20O7 with 13 degrees of unsaturation based on the [M+Na]+ ion at m/z 419.1100 (calcd for C22H20O7Na, 419.1107). The IR absorptions at 1725, 1658, and 1605 cm1 indicated the presence of several carbonyl groups. The 1H NMR spectrum showed resonances for a 1,2,3,4-tetrasubstituted benzene moiety (ring A) at dH 6.83 (1H, d, J = 1.8, H-2) and 6.70 (1H, d, J = 1.8, H-4), a 1,2,3,5-tetrasubstituted benzene ring (ring B) [dH 7.60 (1H, d,

http://dx.doi.org/10.1016/j.phytol.2015.01.006 1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

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Fig. 1. Chemical structures of compounds 1–6 from C. pedunculatum.

(dH 4.64) to C-20 (dC 200.7) and C-30 (dC 144.8), H2-40 (dH 5.89 and 6.16) to C-20 and C-50 (dC 18.5), and H-50 (dH 1.92) to C-20 and C-40 (dC 123.5). Furthermore, the locations of the substituents (two methoxyl, one methoxycarbonyl, and one prenyl) on the xanthone nucleus were also assigned at C-1 (dC 161.6), C-3 (dC 141.7), C-5 (dC 155.5), and C-6 (dC 118.6), respectively, based on correlations observed in the HMBC spectrum (Fig. 2). Accordingly, compound 1 was determined as 1,5-dimethoxy-6-methoxycarbonyl-3-(30 -methyl-20 -oxo-but-30 -enyl)-xanthone and has been given the trivial name comastomaxanthone A. Interestingly, the 3-methyl-2-oxobut-3-enyl moiety is a rare functionality as a substituent of xanthone (Jantan and Saputri, 2012; Nilar and Harrison, 2002). Compound 2 was obtained as a yellow gum. It gave a molecular formula of C21H18O7 according to its HRESIMS at m/z 405.0957 [M+Na]+ (calcd 405.0950). Comparison of the NMR (Table 1) and MS data of 2 with those of 1 demonstrated that 2 had the same skeleton as 1 except for the replacement of the methoxy substituent at C-5 in 1 by a hydroxy group in 2. This structural

J = 8.2, H-7) and 7.41 (1H, d, J = 8.2, H-8)], protons of two methylene groups [dH 5.89 (1H, br s, H-40 a) and 6.16 (1H, br s, H-40 b); dH 4.64 (2H, s, H2-10 )], a methyl group at dH 1.92 (3H, s, H3-50 ) and three methoxy groups at dH 3.80 (3H, s), 3.81 (3H, s), and 4.15 (3H, s). Analyses of the 13C NMR and DEPT data (Table 1), aided by a HSQC experiment, disclosed the presence of three carbonyl groups, nine sp2 quaternary carbons (four of which are oxygen bearing), four sp2 methines, one sp3 methylene, one sp2 methylene, and four methyl groups (including three oxygenated ones). The initial analysis of these NMR spectroscopic data indicated that the molecule consists of a xanthone skeleton (dC 161.6, 109.0, 141.7, 105.3, 155.5, 118.6, 126.5, 121.9, 181.7, 156.6, 124.2, 117.4, and 148.9) (Jantan and Saputri, 2012) with two methoxy groups (dC 56.1 and 61.2), a methoxycarbonyl group (dC 169.0 and 53.0), and a prenylated sidechain (dC 37.6, 200.7, 144.8, 123.5, and 18.5). The HMBC spectrum provided further evidence for the structural assignment (Fig. 2). Especially, the prenyl moiety was established as a rare 3-methyl-2oxo-but-3-enyl substituent by key HMBC correlations from H-10

Table 1 1 H and 13C NMR data for compounds 1–3 (d in ppm, in CDCl3, 500 and 125 MHz). No.

1

2

dC 1 2 3 4 5 6 7 8 9 4a 8a 9a 10a 10 20 30 40 50 60 1-OMe 5-OMe 60 -OMe Ar–OH

161.6 109.0 141.7 105.3 155.5 118.6 126.5 121.9 181.7 156.6 124.2 117.4 148.9 37.6 200.7 144.8 123.5 18.5 169.0 56.1 61.2 53.0

dH (multi., J, Hz) s d s d s s d d s s s s s t s s t q s q q q

6.83 d (1.8) 6.70 d (1.8)

7.60 d (8.2) 7.41 d (8.2)

4.64 s

5.89, 6.16 s 1.92 s 3.80 s 3.81 s 4.15 s

3

dC 161.3 108.3 141.3 105.1 153.9 119.6 126.4 121.5 181.2 156.3 124.2 116.9 150.7 37.4 201.1 144.4 123.1 18.4 169.2 56.0

dH (multi., J, Hz) s d s d s s d d s s s s s t s s t q s q

52.7 q

6.82 d (1.8) 6.69 d (1.8)

7.69 d (8.2) 7.41 d (8.2)

4.63 s

5.85, 6.13 s 1.92 s 3.80 s 4.15 s 10.29 s

dC 161.2 108.1 141.3 105.5 156.9 110.3 155.3 109.0 181.3 156.2 126.9 117.0 142.9 38.2 200.1 145.2 123.9 18.8 169.5 56.0 61.2 52.9

dH (multi., J, Hz) s d s d s s s d s s s s s t s s t q s q q q

6.80 d (1.9) 6.68 d (1.9)

6.98 s

4.61 s

5.84, 6.14 s 1.90 s 3.81 3.82 4.14 10.42

s s s s

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3. Materials and methods 3.1. General experimental procedures

Fig. 2. Selected HMBC (H ! C) correlations of compound 1.

assignment was readily further confirmed by the HMBC corrletaion from the hydroxy proton (dH 10.29) to C-5 (dC 153.9). Thus, the structure of 2 (comastomaxanthone B) was determined to be 5-hydroxy-1-methoxy-6-methoxycarbonyl-3-(30 -methyl-20 - oxobut-30 -enyl)-xanthone. Comastomaxanthone C (3) was assigned the molecular formula C22H20O8 by the positive HRESIMS at m/z 435.1062 [M+Na]+ (calcd 435.1056). The NMR data of 3 were similar to those of 1 except for the ring-B region. The methane group at C-7 in 1 was substituted by an additional hydroxy group in 3, which was proved by the correlations from the hydroxy proton (dH 10.42) to C-7 (dC 155.3) in the HMBC experiment. Therefore, compound 3 was unambiguously determined as 7-hydroxy-1,5-dimethoxy-6-methoxycarbonyl3-(30 -methyl-20 -oxo-but-30 -enyl)-xanthone. The known compounds were identified as isojacereubin (4) (Wu et al., 1998), 1,3,6-trihydroxy-7-methoxy-2,8-di(3-methyl-but-2enyl)-xanthone (5) (Nilar and Harrison, 2002), and 1,6-dihydroxy3,7-dimethoxy-2-(3-methyl-but-2-enyl)-xanthone (6) (Nilar and Harrison, 2002) (Fig. 1), by comparison of their 1D spectroscopic data with those reported in the literature. Since many xanthones have been reported to possess potential anti-virus property (El-Seedi et al., 2010; Wu et al., 2013), compounds 1–6 were tested for their anti-TMV activities. The anti-TMV activities were tested using the half-leaf method (Hu et al., 2013). Ningnanmycin (a commercial product for plant disease in China), was used as a positive control. The results showed that compounds 1–6 all exhibited weak anti-TMV activity with IC50 values ranging from 122.7 to 242.9 mM. (Table 2). The protective effect of 1–6 against TMV was also evaluated by pretreating the tobacco leaves with individual compounds for 6 h before inoculation with TMV. The results (Table 2) showed that at a concentration of 20 mM compounds 1–6 showed protective effects to the host plants, with inhibition rates of 22.1–13.5%, respectively. The results indicated that pretreatment with these xanthones may increase the resistance of the host plant to TMV infection.

UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on a Bruker DRX-500 instrument with TMS as the internal standard. Unless otherwise specified, chemical shifts (d) were expressed in ppm with reference to the solvent signals. Mass spectra were performed on a VG Autospec-3000 spectrometer under 70 eV. Column chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China). Lichroprep RP-18 gel (40–63 mm, Merck, Darmstadt, Germany), and MCI gel (75–150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm  25 cm, cm, column. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. 3.2. Plant material The aerial parts of Comastoma pedunculatum were collected in Shangri-la Prefecture, Yunnan Province, People’s Republic of China, in August 2012. The identification of the plant material was verified by Dr. N. Yuan of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (YNNI-2012-20) has been deposited in our laboratory. 3.3. Extraction and isolation The air-dried and powdered aerial parts of C. pedunculatum (4.6 kg) were extracted four times with 80% aqueous acetone (3  10 L) at room temperature and filtered. The extract was partitioned between EtOAc and H2O. The EtOAc fraction (185 g) was chromatographed on MCI gel CHP 20P eluted with 90:10 MeOH/H2O. Then, the 90% MeOH fraction (125 g) was submitted to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3-acetone gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. Fraction B (9:1, 22 g) was subjected to silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures B1–B5. Fraction B2 (8:2, 3.5 g) was subjected to preparative HPLC (60% MeOH, flow rate 12 mL/ min) to give 1 (10.1 mg), 2 (6.2 mg), and 3 (12.5 mg). Fraction B3 (7:3, 5.6 g) was further chromatographed over RP-18 column followed by semi-preparative and preparative HPLC to give 4 (2.8 mg), 5 (13.2 mg), and 6 (1.9 mg). 3.4. Anti-TMV assays

Table 2 Anti-TMV activity of compounds 1–6 on Nicotiana tabacum leaf, and protective effect on TMV infection. Compounds

Inhibition rates at 20 mM (%)a

IC50 (mM)a

Inhibition rates at 20 mM (%)b

1 2 3 4 5 6 Ningnamycin

22.2  2.2 16.7  2.4 18.5  2.8 23.2  2.4 19.5  2.6 14.6  2.2 30.5  2.8

138.5 186.2 157.9 122.7 167.4 242.9 52.4

20.6  2.5 14.7  2.7 13.5  2.2 22.1  2.6 21.7  2.4 13.5  2.9 28.6  3.2

a b

Anti-TMV activity. Protective effect on TMV infection.

TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, PR China. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as described. The concentration of TMV was determined as 20 mg/mL with a UV spectrophotometer 260 nm ½Virus concentration ¼ ðA260  dilution ratioÞ=E10:1%; . The pucm rified virus was kept at 20 8C and diluted to 32 mg/mL with 0.01 M PBS before use. Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. N. glutinosa was used as a local lesion host. The experiments were conducted when the plants grew to the 5–6-leaf stage. The tested compounds were dissolved in DMSO and diluted with distilled H2O to the required concentrations. A solution of equal concentration of DMSO was used as a negative control. The

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commercial antiviral agent ningnanmycin was used as positive control. For the half-leaf method, the virus was inhibited by mixing with the solution of compound. After 30 min, the mixture was inoculated on the left side of the leaves of N. glutinosa, whereas the right side of the leaves was inoculated with the mixture of DMSO solution and the virus as control. The local lesion numbers were recorded 3 or 4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula:   ðC  TÞ  100% inhibition rate ð%Þ ¼ C where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment. 3.5. Spectroscopic data Comastomaxanthone A (1), C22H20O7, yellow gum; UV (MeOH)

lmax (log e) 210 (4.15), 246 (3.52), 315 (3.85) nm; IR (KBr) nmax 3070, 2928, 2870, 1725, 1658, 1605, 1542, 1476, 1357, 1120, 1049, 853, 784 cm1; 1H and 13C-NMR spectroscopic data (CDCl3, 500 and 125 MHz), see Table 1; ESIMS m/z (positive ion mode) 419 [M+Na]+; HRESIMS (positive ion mode) m/z 419.1100 [M+Na]+ (calcd for C22H20O7Na, 419.1107). Comastomaxanthone B (2), C21H18O7, yellow gum, UV (MeOH) lmax (log e) 210 (4.04), 242 (3.47), 312 (3.76) nm; IR (KBr) nmax 3438, 3059, 2921, 2864, 1722, 1654, 1602, 1562 1473, 1359, 1132, 1055, 861, 792 cm1; 1H and 13C-NMR spectroscopic data (CDCl3, 500 and 125 MHz), see Table 1; ESIMS m/z (positive ion mode) 405 [M+Na]+; HRESIMS (positive ion mode) m/z 405.0957 [M+Na]+ (calcd for C21H18O7Na, 405.0950). Comastomaxanthone C (3), C22H20O8, yellow gum, UV (MeOH) lmax (log e) 210 (4.16), 248 (3.69), 315 (3.82) nm; IR (KBr) nmax 3438, 3059, 2914, 2857, 1718, 1650, 1608, 1549, 1436, 1378, 1146, 1072, 872, 765 cm1; 1H and 13C-NMR spectroscopic data (CDCl3, 500 and 125 MHz), see Table 1; ESIMS m/z (positive ion mode) 435 [M+Na]+; HRESIMS (positive ion mode) m/z 435.1062 [M+Na]+ (calcd for C22H20O8Na, 435.1056).

Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 21262048), and the program for Innovative Research Team (in Science and Technology) in University of Yunnan Provinve (NO. IRTSTYN 2014-11).

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